Interferons (IFNs) are a group of naturally-produced pleiotropic glycoproteins, known as cytokines, secreted from different kinds of cells (epithelial cells, fibroblasts, lymphocytes, macrophages) by induction from a series of stimulations (virus, bacteria, cells, tumours and macromolecules) and endowed with antiviral, anti-proliferative, and immuno-modulatory properties as well as an analgesic action. Following its endogenous production or its administration, the interferon interacts with the specific receptors on the cells surface and starts the transduction of the signal through the cytoplasm until the nucleus, inducing the expression of genes which codify for specific proteins having antiviral and immuno-stimulating activity. The medical potential of IFNs has been recognized, as demonstrated by the approval of some different types of IFNs for use in humans, such as IFN1a (Rebif, Avonex), IFN1b (Betaseron), as drugs for the treatment of multiple sclerosis, and recombinant human IFNa2a (Roferon A) and IFNa2b (Intron A), as drugs for the treatment of malignant (cancer) and viral diseases.
Based on the type of receptor through which IFNs signal, human IFNs have been classified into three major types, namely, (i) IFN type I [all type I IFNs bind to a specific cell surface receptor complex known as the IFN-alpha receptor (IFNAR)—type I IFNs in humans are IFN alpha (IFN-α), IFN beta (IFN-β) and IFN omega (IFN-ω)], (ii) IFN type II [type II IFN binds to a IFN-gamma receptor (IFNGR)—type II IFN in humans is IFN gamma (IFN-γ)], and IFN type III [type III IFNs signal through a receptor complex consisting of IL10R2 and IFNLR1]. IFNs alpha and beta, known as type I IFNs, are structurally correlated, stable at acid pH and compete for the same cell-receptor (IFNAR).
At present, IFNs alpha, beta and gamma can be manufactured under recombinant form with the double advantage of getting much higher amounts of product compared to those obtained through the isolation from natural sources (leukocytes, fibroblasts, lymphocytes) and of reducing the complexity of the processes of purification and check of the safety of the product. In fact, most of the marketed pharmaceutical grade recombinant IFN is produced and purified from Escherichia coli. 
The E. coli recombinant protein expression system has been, and still is, the system of choice for the production of IFN. Indeed, IFN genes do not have introns, and the protein products are generally not glycosylated. Furthermore, E. coli can grow rapidly to high cell densities, and strains used for recombinant protein production have been genetically modified so that they are generally regarded as safe for large-scale fermentation.
The expression of IFN cDNA was achieved directly in E. coli soon after it was first cloned [Goedell et al. Nature., 287, 411-416, 1980; Pestka, S. Arch. Biochem. Biophys., 221 (1), 1-37, 1983; Mizoguchi et al. DNA., 4, 221-32, 1985; Pestka et al. Ann. Rev. Biochem., 56, 727-777, 1987; Baron and Narula. Critical reviews in Biotechnology, 10 (3), 179-190, 1990]. In fact, IFN alpha (IFNa) has been one of the first proteins to be produced by means of E. coli with the DNA recombinant technology [Derynck et al., Nature, 287, 193-197, 1980; Nagata et al., Nature, 284, 316-320, 1980].
However, the expression of IFNs in E. coli shows some problems. IFNs expressed in large amount in E. coli often precipitate into insoluble aggregates called inclusion bodies (IBs) [Swaminathan et al., Prot Express. Purif., 15, 236-242, 1999; Bedarrain et al., Biotechnol. Appl. Biochem., 33, 173-182, 2001; Srivasta et al. Prot. Express. Purif 41, 313-322, 2005] that are, in general, misfolded proteins and thus biologically inactive [Villaverde and Carrio, Biotechnol. Lett., 25, 1385-1395, 2003]. To get such proteins under native form it is necessary to submit said IBs to a denaturation phase followed by a renaturation phase, oxidizing, in case that disulfide bridges have to be formed as in the natural protein. Further, the incorporation of an extra methionine residue at the N-terminal end of the target protein sequence (e.g., an IFN) is a characteristic feature of protein expression in E. coli. As it is known, methionine residues distributed within the sequence of a protein are prone to oxidation. Such process may occur during the process for producing the protein or the pharmaceutical composition comprising said protein (if it can be used as a drug, e.g., an IFN) and is more pronounced during long-term storage at elevated temperatures.
Although several methods of production and purification of IFNs in bacteria as IBs have been developed [e.g., Thatcher and Panayotatos, Methods Enzymol. 119, 166-177, 1986; U.S. Pat. No. 4,511,502; U.S. Pat. No. 4,765,903; U.S. Pat. No. 4,845,032; EP 1310559; or EP 1990349], there are further factors which may present obstacles for successful production and purification of IFNs, namely, an IFNa of therapeutical degree, such as the incorporation of an extra methionine residue at the N-terminus of the target IFN and the generation of its oxidised species which have to be removed (if the product is to be used as a drug) thus reducing the overall yield in the production of IFN, increasing the complexity of the purification process and rendering the process for production and purification of alpha IFNs of therapeutical degree in a laborious process.
Accordingly, there remains a need for a method that enables the production of IFNa, particularly, IFNa5, of therapeutical degree from E. coli host cells in a high yielding and cost-effective manner.